Method for producing purified recombinant Haemophilus influenzae transferrin binding proteins

Purified and isolated nucleic acid is provided which encodes a transferrin receptor protein of a strain of Haemophilus or a fragment or an analog of the transferrin receptor protein. The nucleic acid sequence may be used to produce peptides free of contaminants derived from bacteria normally containing the Tbp1 or Tbp2 proteins for purposes of diagnostics and medical treatment. Furthermore, the nucleic acid molecule may be used in the diagnosis of infection. Also provided are recombinant Tbp1 or Tbp2 and methods for purification of the same. Live vectors expressing epitopes of transferrin receptor protein for vaccination are provided.

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1. A method of producing an isolated and purified Tbp1 or Tbp2 protein of a strain of Haemophilus influenzae, comprising the steps of:

(a) providing a recombinant host expressing, in inclusion bodies, a Tbp1 or Tbp2 protein, but not both, of a strain of Haemophilus influenzae;
(b) growing said host to provide a cell mass wherein said Tbp1 or Tbp2 protein is present in inclusion bodies;
(c) disrupting the cell mass to provide a cell lysate;
(d) fractionating the cell lysate to provide a first supernatant and a first pellet, the first supernatant comprising substantially a large proportion of soluble host proteins;
(e) separating said first supernatant from said first pellet;
(f) selectively extracting the first pellet to remove substantially all soluble host proteins and host membrane proteins therefrom to provide a second supernatant and an extracted pellet containing the inclusion bodies;
(g) separating said second supernatant from said extracted pellet;
(h) solubilizing the extracted pellet to provide a solubilized extract; and
(i) fractionating the solubilized extract to provide a Tbp1 or Tbp2 protein containing fraction.

2. The method of claim 1 wherein the cell lysate is fractionated by centrifugation thereof.

3. The method of claim 2 wherein the step of selectively extracting the first pellet comprises at least one detergent extraction.

4. The method of claim 3 wherein the solubilized extract is fractionated by gel filtration to provide said Tbp1 or Tbp2 protein containing fraction.

5. The method of claim 4 including subsequently dialyzing the Tbp1 or Tbp2 protein containing fraction to remove at least said detergent to provide a further purified solution of Tbp1 or Tbp2 protein.

Referenced Cited
U.S. Patent Documents
5141743 August 25, 1992 Schryvers
Foreign Patent Documents
9308283 April 1993 CAX
Other references
  • Ferron, et al 1993. FEMO Microbiol. Letters 109: 159-166. Schryvers, 1989. J. Med. Microbiol. 29: 121-130. Sofer, et al. 1983. BioTechniques, "Designing an Optimal Chromatographic Purification Scheme for Proteins". Stevenson, et al. 1992. Infect. and Immun. 60(6):2391-96. Cornelissen, et al. 1992. J. Bact. 174(18): 5788-97.
Patent History
Patent number: 5708149
Type: Grant
Filed: Jun 7, 1995
Date of Patent: Jan 13, 1998
Assignee: Connaught Laboratories Limited (North York)
Inventors: Sheena Loosmore (Aurora), Robin Harkness (Willowdale), Anthony Schryvers (Calgary), Pele Chong (Richmond Hill), Scott Gray-Owen (Calgary), Yan-Ping Yang (Willowdale), Andrew Murdin (Newmarket), Michel Klein (Willowdale)
Primary Examiner: Nancy Degen
Assistant Examiner: Matthew Latimer
Law Firm: Sim & McBurney
Application Number: 8/487,890